This invention relates generally to biology and more particularly concerns the process of preserving biological materials by freezing.
Interest in cryobiological research has been recorded for approximately 200 years. Since 1949, when glycerol was introduced as a freeze-thaw protective agent, research in this field has experienced a significant upsurge.
This upsurge in activity is largely due to the improved success-failure ratios afforded by the use of glycerol.
One theory advanced to explain the success experienced with glycerol is that increased salt concentration during the freezing process causes damage to the biological material, and that glycerol acts as a salt buffer. A parallel theory, however, is that ice crystals formed during the freezing process damage the biological material by compressing, puncturing or disarranging it, and the possibility of such mechanical ice damage increases with the size of the crystals formed. Glycerol is therefore considered effective against such damage because it modifies intra and extra cellular ice crystallization in the biological material.
Considering this problem of mechanical damage caused by ice crystallization from a slightly different perspective, not only temperature but also the duration of the freezing cycle bears significance. For example, it is believed that a fast freezing cycle results in the formation of smaller ice crystals and therefore minimizes damage. On the other hand, it is also proposed that a slow freezing cycle is conducive to greater dehydration of the biological material which in turn minimizes damage. While these theories appear to be in conflict, each may be applicable depending on the particular biological material involved.
Many other factors have been found to related to the success-failure ratio of the freezing process. Among them is the phenomenon of temperature shock, also called cold or thermal shock, in which rapid temperature change causes damage in different biological materials due to alteration of various physiological functions in the materials. Also, minimum mass and maximum surface area of the biological material to be frozen are found to enhance the degree of temperature control.
The foregoing discussion is given to illustrate the factors related to the success of the freezing process. It is seen that regardless of the biological material being frozen, or of the cryobiological theory applied, the success of the freezing process will be largely dependent on the accurate control of the temperature of the biological material and the rate of the change of that temperature throughout the freezing process. Therefore, in order to understand the problems with which the present invention deals, the freezing process itself should be at least briefly examined.
The freezing cycle may be considered in three separate stages. The first stage, during which the biological material is in the liquid phase, extends from some initial temperature, perhaps ambient room temperature, to the freezing temperature of the biological material. This initial temperature is merely a point of reference at which the cycle may be considered to begin and is not critical. The rate of cooling and the final temperature of this stage are critical, however. Rapid changes in temperature may cause damage to the biological material, as suggested in the temperature shock theory. Also, a substantially linear approach to the freezing temperature assures maximum temperature control at that point. Reaching the exact freezing temperature or a temperature minimally above it as the terminal point of the first stage is critical because, if the second, or phase change, stage is initiated at any temperature appreciably higher than the freezing temperature, rapid temperature drop will occur, possibly causing temperature shock. On the other hand, if the first stage is not concluded at freezing temperature, the delay will cause supercooling of the biological material and a rapid return to freezing temperature when phase change is initiated, also possibly causing temperature shock.
The second stage of the freezing process preferably takes place at a constant biological material temperature and extends from the point at which the biological material reaches freezing temperature to the point at which the latent heat of fusion has been removed. As has been pointed out, the completion of the first and initiation of the second stage at or slightly above the freezing point of the biological material is essential to avoid either supercooling of or temperature shock to the material. Moreover, it is during this second phase that crystallization occurs. Therefore, it is necessary that the rate at which the latent heat of fusion is removed be accurately controlled, so that damage resulting from too slow or too rapid crystallization can be avoided. Furthermore, it is essential that the second stage be concluded at the point at which the latent heat of fusion is removed because, since extremely low chamber temperatures are required in this stage, continuance beyond this point may also cause temperature shock to the biological material.
The third and final stage of the freezing process extends from the point at which the latent heat of fusion has been removed to a preselected final temperature at which the biological material is to be stored. Again it is necessary, to avoid the damage of temperature shock, that there be a substantially linear approach to this final temperature.
Various methods and apparata have been devised to accomplish the control of temperature and rate of change of temperature during the freezing process. Among these is the temperature differential method, according to which differential thermocouples sense the temperature difference between a dummy sample and the cooling chamber. If this difference drops below a preselected value, more coolant is demanded by the system until the differential reaches the preselected value. This method is workable, but has several shortcomings. Maintenance of the same temperature difference throughout the freezing process results in different cooling rates in each of the process stages. Increasing or decreasing the preselected difference to achieve desirable results in one of the stages may well produce undesireable results in either or both of the other stages. Also, since temperature difference rather than the temperature of the dummy sample is controlling, supercooling will occur at the end of the first stage. Furthermore, since the size and geometry of the sample vary its cooling rate, preselection of the most appropriate temperature difference for a given process application can cause difficulty.
Another method of controlling the freezing process is known as the cam type program, according to which the temperature of the biological material is forced to follow a preselected temperature curve through use of some type of comparator circuit. This method provides more stage to stage flexibility than the temperature differential method, but introduces other problems. For example, any change in sample size, geometry, material, protective additive, and so on, will require a different preselected temperature curve. This problem is compounded by the fact that, since introduction of coolant to the sample is triggered via comparison to the preselected curve, the sample temperature does not exactly follow that curve but rather will fluctuate between the curve and some maximum differential above or below it. This method is therefore one of trial and error to produce a curve providing a desired result for a particular sample of given size, geometry and material.
One interesting method of accomplishing rapid phase change of the biological material contemplates precompression of the material and exposure to low temperature. On decompression the material therefore solidifies much more rapidly than if it were exposed to the same temperature in a nonprecompressed state. However, the method deals only with the phase change stage of the freezing process, and control of temperature change rates, if any, would be reliant on accurate mechanical control of the application and release of pressure.
Accordingly, it is an object of this invention to preserve biological materials by freezing.
It is a further object of this invention to minimize the risk of supercooling the biological material prior to the initiation of phase change.
Another object of the invention is to minimize the risk of temperature shock to the biological material.
A correlated object of the invention is to permit preselection of cooling rates, duration of phase change, and storage temperature.